Design, synthesis and biological evaluation of novel arylidine- malononitrile derivatives as non-carboxylic inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1007/s00044-013-0528-1

In this study, we describe the design, synthesis, biological evaluation and molecular modelling studies of novel non-carboxylic arylidine malononitrile-based molecules as Protein Tyrosine Phosphatase 1B (PTP1B) inhibitors. The synthesized derivatives were evaluated in vitro for glucose reuptake using L6 muscle cell lines and enzymatic assay against PTP1B. The biological activity results showed that the 2-methoxy substituted (14b) compound exhibited significant activity in both the assays. The unsubstituted compound (14a) also possessed comparable activity on glucose reuptake in L6 muscle cell lines and better inhibitory activity on PTP1B enzyme assays. Docking analysis was performed on the most potent compound of the series to understand the nature of interactions governing the binding of the designed molecule with the PTP1B enzyme.

Full text of DOI:10.1007/s00044-013-0528-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0528-1
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of novel arylidine-
malononitrile derivatives as non-carboxylic inhibitors
of protein tyrosine phosphatase 1B
•
Girdhar Singh Deora Chandrabose Karthikeyan N. S. Hari Narayana Moorthy
•
•
•
Vandana Rathore Arun K. Rawat Akhilesh K. Tamrakar
•
•
•
A. K. Srivastava Piyush Trivedi
Received: 15 August 2012 / Accepted: 31 January 2013
Ó Springer Science+Business Media New York 2013
Abstract In this study, we describe the design, synthesis,
biological evaluation and molecular modelling studies of
novel non-carboxylic arylidine malononitrile-based mole-
cules as Protein Tyrosine Phosphatase 1B (PTP1B) inhib-
itors. The synthesized derivatives were evaluated in vitro
for glucose reuptake using L6 muscle cell lines and enzy-
matic assay against PTP1B. The biological activity results
showed that the 2-methoxy substituted (14b) compound
exhibited significant activity in both the assays. The
unsubstituted compound (14a) also possessed comparable
activity on glucose reuptake in L6 muscle cell lines
and better inhibitory activity on PTP1B enzyme assays.
Docking analysis was performed on the most potent com-
pound of the series to understand the nature of interactions
governing the binding of the designed molecule with the
PTP1B enzyme.
Keywords PTP1B Á Diabetes Á Arylidine malononitrile Á
Oxadiazole Á Non-carboxylic acid inhibitors Á
Docking study
Introduction
Diabetes mellitus is a major metabolic syndrome affecting
human health in developing and developed nations. There
are an estimated 285 million adults with diabetes in 2010
and this number will continue to increase globally to affect
300 million people by the year 2025 due to an ageing
population, growth of population size, urbanization and
high prevalences of obesity and sedentary lifestyle (Zhang
et al., 2010; Simpson et al., 2003; Shaw et al., 2010; Dixit
et al., 2007). Diabetes leads to both premature death and
complications such as blindness, amputations, renal disease
and cardiovascular diseases (Zhang et al., 2010; Egede and
Ellis, 2010; Nicholas, 2003).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0528-1) contains supplementary
material, which is available to authorized users.
G. S. Deora Á C. Karthikeyan Á N. S. H. N. Moorthy Á
P. Trivedi (&)
School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki
Vishwavidyalaya, Airport Bypass Road, Gandhi Nagar,
Bhopal 462036, MP, India
e-mail: drtrivedislab@gmail.com; piyush.trivedi@rgtu.net
G. S. Deora Á V. Rathore
Dr. Reddy’s Institute of Life Sciences, University of Hyderabad
Campus, Gachibowli, Hyderabad 500046, India
Protein tyrosine phosphatase 1B (PTP1B) has been the
focus of antidiabetic drug discovery efforts owing to evi-
dence of its role in downregulating insulin transduction
mediated by receptor tyrosine kinases such as insulin
receptor and epidermal growth factor receptor (Johnson
et al., 2002; Cheon et al., 2004). Inhibitors of this enzyme
are predicted to enhance insulin-stimulated glucose trans-
port and have potential for the treatment of type II diabetes.
Thus, there is a great interest in PTPs as molecular targets
for the development of novel therapeutic agents (Cheon
et al., 2004).
N. S. H. N. Moorthy
Department of Chemistry and Biochemistry, Faculty of Sciences,
University of Porto, 687, Rua de Campo Alegre,
4169-007 Porto, Portugal
V. Rathore
B. N. Institute of Pharmaceutical Sciences, Udaipur 313001,
Rajasthan, India
A. K. Rawat Á A. K. Tamrakar Á A. K. Srivastava
Division of Biochemistry, Central Drug Research Institute,
Lucknow 226001, UP, India
123

Med Chem Res
In last few years, large numbers of potent and selective
PTP1B inhibitors have been reported in the literature (Pei
et al., 2003; Xin et al., 2003; Zhao et al., 2004; Cho et al.,
2006; Wan et al., 2006). Most of the inhibitors reported
incorporate a charged pTyr mimetic group such as phos-
phonates, carboxylic acids and sulphamic acids since the
active site of enzyme accommodates pTyr, which contains
two negative charges at physiological pH. Consequently,
the molecules are generally not drug-like and they possess
limited cell membrane permeability and bioavailability.
Hence, small molecule PTP1B inhibitors devoid of any
charged moieties and with good enzyme binding affinity is
highly desirable.
chromatography. The final step of synthesis included a simple
Knoevenagel condensation of substituted aldehydes with mal-
ononitrile (Please refer Supplementary information for detailed
synthetic procedure).
The effect of synthesized compounds 14a–h on protein
tyrosine phosphatase inhibition was studied using colori-
metric, non-radioactive PTP1B tyrosine phosphatase drug
discovery kit-BML-AK 822 from Enzo Life Sciences,
USA (Joshi et al., 2012a, b). The synthesized compounds
14a–h were also evaluated for glucose reuptake using L-6
muscle cell lines (Table 1). The percentage glucose reuptake
results showed that the compound with 2-methoxy substi-
tution in the aromatic ring (14b) possessed significant
activity than any other compounds in the series. Introduction
of nitro group (14 h) or electronegative fluoro group (14f) in
the aromatic ring resulted in decreased glucose reuptake
activity. Further, substitution in the meta position on the
aromatic ring (14c and 14h) appears to be unfavourable to
glucose reuptake activity shown by the title compounds. The
unsubstituted derivative (14a) showssignificant activity than
meta- and para-substituted compounds (14c, 14e–h) but
these compounds (14c, 14e–h) exhibit comparable activity
with the reference compound (rosiglitazone). Overall, the
SAR study suggests that substitution in ortho position
confers greater glucose uptake activity.
Numerous reports on development of non-carboxylic
PTP1B inhibitors have emerged recently in literature.
Scientists have used many parent structures for novel
molecules development, among them, thiazolidinedione
derivatives (1) (Bhattarai et al., 2009), barbituric acid
derivatives (2) (Kafle et al., 2011), isothiazolidinone inhib-
itors (3) (Douty et al., 2008), chromones derivatives (4)
(Forghieri et al., 2009) and pyridazine analogues (5) (Lilje-
bris et al., 2002) possessed significant activity (Fig. 1). As
part of our efforts to discover novel non-carboxylic acid
PTP1B inhibitors, we identified arylidine malononitrile as a
suitable phosphate mimic through computer-aided drug
design protocol involving docking analysis. The arylidene
malononitrile moiety exhibits favourable interactions with
residues flanking the pTyr in the active site of PTP1B and is
also well suited for further chemical derivatization. To the
best of our knowledge, inhibitory activity of this class of
compounds against phosphatases has not been reported in the
literature. The most potent compounds were also evaluated
using zebrafish embryos for general toxicity related effects at
10 lM concentration. Developing embryos of the zebrafish
(Danio rerio) are excellent animal models for studying
specific developmental effects of small molecules, thus
allowing effective in vivo evaluations of potential drugs
before embarking on highly expensive studies on rodents and
humans.
The PTP1B inhibitory activity results show that the
unsubstituted compound (14a) possessed significant activ-
ity than other compounds in the series (14b–h). Together
with the good glucose reuptake activity, the compound 14b
also possessed better PTP1B inhibitory activity than other
compounds in the series (14c–h). The 2-chloro-substituted
compounds (14d) exhibited the least activity in the series.
In contrast to the glucose reuptake activity, the compounds
substituted on the 3-position of the benzene ring (14c and
14h) exhibited significant PTP1B inhibitory potency.
The biological activity results showed that the substi-
tution on the benzene ring connected directly to the oxa-
diazole ring has little effect on enhancing PTP1B inhibition
but elicits greater effect in glucose reuptake activity. It is
confirmed from the activity results from both assays that
the unsubstituted compound (14a) has remarkable activity.
Even though the meta-substitution (14c and 14h) on the
benzene ring is detrimental for the glucose reuptake
activity it is favoured for the PTP1B inhibition. Both
electron releasing substituents such as methyl and methoxy
and electron withdrawing substituents in the molecules
exhibit enhanced PTP1B inhibitory activity, while elec-
tronegative halogen groups (14d–f) were found to be
unfavourable for the activity. Moreover, the lack of cor-
relation between the glucose uptake assay results and
PTP1B inhibition assay result highlights that multiple
mechanisms may be involved in the antidiabetic potency
showed by the studied compounds. Nevertheless, the
Results and discussion
Substituted 2-{4-[2-(5-phenyl-1,3,4-oxadiazol-2-ylthio) ethoxy]
benzylidene} malononitrile-based derivatives (14a–h) were
synthesized by known synthetic approaches in several stages,
which included reactions of esterification, cyclization and con-
densation (Scheme 1). Substituted oxadiazoles (10a–h) were
synthesized in three steps according to reported procedure
(Zarghi et al., 2008) from the appropriate benzoic acids (7a–h).
The linker moiety (12) was synthesized using para-hydroxy-
benzaldehyde (11) and 1,2 di-bromoethane by reported proce-
dure (Kumar et al., 2007) and purified by column
123

Med Chem Res
Fig. 1 Structure of potent
PTP1B inhibitors and
compounds synthesized in the
study
Scheme 1 Reagent and
conditions: a EtOH, reflux, 8 h;
b NH2NH2.H2O, reflux, 6 h; c
CS2, Ethanol, reflux, 5 h; d 1,2-
di-bromoethane, K2CO3,
acetone, 70 °C, 20 h; e K2CO3,
acetone, 70 °C, 2–4 h; f
malononitrile, methanol, R.T.,
1–2 h
PTP1B inhibition showed by the compounds and the
novelty of the structure in comparison to the previously
reported PTP1B inhibitors warrant further in silico studies
to understand their binding mode to PTP1B enzyme.
Related to the foregoing, molecular docking studies were
performed in order to gain further insight into the binding mode
of the compounds into PTP1B enzyme. The most potent
compounds of the series were docked into the active site of the
enzyme using using Maestro, version 9.2 implemented from
Schrodinger software suite (Maestro, 2011). The protein for
docking study was obtained from Protein Data Bank (PDB ID:
1Q1M), was prepared by removing solvent, adding hydrogens
and minimization in the presence of bound ligand using protein
preparation wizard. Grids for molecular docking were gener-
ated with bound co-crystallized ligand. The compounds were
docked using Glide in extra-precision (XP) mode, with up to
three poses saved per molecule.
The docking study has predicted good binding of the
compounds with the catalytic binding site of PTP1B. The
Docking orientation and interactions of molecule 14a with
catalytic site residues of PTP1B are shown in Figs. 2 and 3.
As expected, the arylidine-malononitrile moiety extends
towards the arginine binding region (SITE A) and the
nitrile group interacts with Arg221 residue in the active site
through hydrogen bonds (Fig. 3). Further, the phenyl ring
of the arylidine malononitrile shows hydrophobic interac-
tion with phenyl ring of Tyr46. In addition to the PTP1B
catalytic site, Puius et al., (1997) has reported an additional
aryl phosphate-binding site in PTP1B. This second aryl
phosphate-binding site is made up of residues such as
123

Med Chem Res
Table 1 In vitro glucose uptake and PTP1B enzyme inhibitory activity for compounds 14a–h
N
O
N
O
N
R
S
N
Compound
R
% PTP1B inhibition (at 10 lM)
% glucose uptake in L6 muscle cell line (at 50 lM)
14a
H
54.6
50.0
39.0
23.4
34.3
30.4
44.5
41.4
–
25.3
38.5
13.5
26.6
24.2
13.3
18.8
11.8
30.2*
25.2**
14b
2-MeO
3-MeO
2-Cl
4-Cl
4-F
14c
14d
14e
14f
14g
4-CH3
3-NO2
–
14h
Metformin
Rosiglitazone
–
–
* At 500 lM
** At 100 lM
Fig. 2 Docking orientation of 14a in catalytic pocket (Site A & B) of
PTP1B
Fig. 3 Binding pose and interactions of 14a with catalytic site
residues of PTP1B
Arg24, Arg254, Met258, Phe52 and Gln262. The nitrogen
atom (N3) of 1,3,4-oxadiazole ring is interacting with
Gln262 of additional phosphate-binding site by hydrogen
bonding as well as few hydrophobic interactions between
substituted phenyl ring and hydrophobic residues of this
additional phosphate-binding site are also observed. Our
special interest was potential interactions between inhibitor
functionality with residues in this additional aryl phos-
phate-binding site, as it has been proven to contribute
towards PTP1B selectivity. By design, it was expected that
the phenyl oxadiazole moiety would be accommodated in
the additional aryl phosphate-binding site (SITE B) and
this orientation would be helped by flexible diethylene
linker. The docking studies show that this orientation can
exist where the phenyl ring attached oxadiazole is flanked
by hydrophobic residues such as Phe52 and Met258.
The most active compounds 14a and 14b were screened
for general toxicity studies using zebrafish embryos. The
embryos were grown in the presence of the compounds for
24, 48 and 72 h. Control embryos were incubated in 0.1 %
DMSO. Finally, embryos were observed using visible light
microscopy at regular intervals after compound treatment
to document general toxicity related effects such as
123

Med Chem Res
molecular modeling of a new series of chromones as low
molecular weight protein tyrosine phosphatase inhibitors. Bioorg
Med Chem Lett 17:2658–2672
developmental delays, deformations, oedema and death.
The results indicate that none of the tested compounds
were found to be having significant toxic effect on zebra-
fish embryos (please refer supplementary information for
detailed experimental procedure).
Johnson TO, Ermolieff J, Jirousek MR (2002) Protein tyrosine
phosphatase 1B inhibitors for diabetes. Nat Rev Drug Discov
1:696–709
Joshi P, Deora GS, Rathore V, Tanwar OP, Rawat AK, Srivastava
AK, Jain D (2012a) Identification of ZINC02765569: a potent
inhibitor of PTP1B by vHTS. Med Chem Res. doi:10.1007/
s00044-012-0007-0
Conclusion
Joshi P, Deora GS, Rathore V, Rawat AK, Srivastava AK, Jain D (2012b)
Molecular modeling and synthesis of ZINC02765569 derivatives as
protein tyrosine phosphatase 1B inhibitors: lead optimization study.
Med Chem Res. doi:10.1007/s00044-012-0165-0
Kafle B, Bhattarai BR, Cho H (2011) Barbituric acid derivatives as
protein tyrosine phosphatase inhibitors. Bull Korean Chem Soc
32:31–32
Kumar R, Mittal A, Ramachandran U (2007) Design and synthesis of
6-methyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid
derivatives as PPARc activators. Bioorg Med Chem Lett 17:4613–
4618
Liljebris C, Martinsson J, Tedenborg L, Williams M, Barker E, Duffy
JES, Nygren A, James S (2002) Synthesis and biological activity
of a novel class of pyridazine analogues as non-competitive
reversible inhibitors of protein tyrosine phosphatase 1B
(PTP1B). Bioorg Med Chem Lett 10:3197–3212
In conclusion, this work reports novel arylidine-malono-
nitrile derivatives as non-carboxylic inhibitors of protein
tyrosine phosphatase 1B. Compound 14a, the unsubstituted
derivative showed maximum PTP1B inhibition whereas the
compound 14b, the 2-methoxy substituted derivative
exhibited maximal efficacy in glucose reuptake assay.
Molecular docking studies of the compound 14a showed
that arylidine-malononitrile group in the compounds inter-
acts with the PTP1B catalytic site and the phenyl oxadiazole
moiety in the compounds is accommodated in the additional
aryl phosphate-binding site. Overall, the present research
work has clearly established the studied arylidene malon-
onitrile derivatives as potential non-carboxylic protein
tyrosine phosphatase 1B inhibitors. Further refinement of
analogues is ongoing and will be reported in due course.
¨
Maestro (2011) version 9.2, Schrodinger, LLC, New York, NY
Nicholas K (2003) PTP1B: from the sidelines to the front lines! FEBS
Lett 546:140–148
Pei Z, Li X, Liu G, Bad-Zapatero C, Lubben T, Zhang T, Ballaron SJ,
Hutchins CW, Trevillyan JM, Jirousek MR (2003) Discovery
and SAR of novel, potent and selective protein tyrosine
phosphatase 1B inhibitors. Bioorg Med Chem Lett 13:3129–
3132
Puius YA, Zhao Y, Sullivan M, Lawrence DS, Almo SC, Zhang ZY
(1997) Identification of a second aryl phosphate-binding site in
protein-tyrosine phosphatase 1B: a paradigm for inhibitor
Acknowledgments The authors gratefully acknowledge SAIF,
Punjab University, Chandigarh for NMR spectral analysis of the
synthesized compounds. The author Girdhar Singh Deora wishes to
thank AICTE, New Delhi for postgraduate fellowship. C. Karthikeyan
is a recipient of a senior research fellowship of CSIR, New Delhi.
design. Proc Natl Acad Sci 94:13420–13425
Shaw JE, Sicree RA, Zimmet PZ (2010) Global estimates of the
prevalence of diabetes for 2010 and 2030. Diabetes Res Clin
Pract 87:4–14
References
Simpson RW, Shaw JE, Zimmet PZ (2003) The prevention of type 2
diabetes—lifestyle change or pharmacotherapy? A challenge for
the 21st century. Diabetes Res Clin Pract 59:165–180
Wan ZK, Lee J, Xu W, Erbe DV, Joseph-McCarthy D, Follows BC,
Zhang YL (2006) Monocyclic thiophenes as protein tyrosine
phosphatase 1B inhibitors: capturing interactions with Asp48.
Bioorg Med Chem Lett 16:4941–4945
Xin Z, Liu G, Bad-Zapatero C, Pei Z, Szczepankiewicz BG, Li X,
Zhang T, Hutchins CW, Hajduk PJ, Ballaron SJ, Stashko MA,
Lubben TH, Trevillyan JM, Jirousek MR (2003) Identification of
a monoacid-Based, cell permeable, selective inhibitor of protein
tyrosine phosphatase 1B. Bioorg Med Chem Lett 13:3947–3950
Zarghi A, Hamedi S, Tootooni F, Amini B, Sharifi B, Faizi M, Abbas
Tabatabai S, Shafiee A (2008) Synthesis and pharmacological
evaluation of new 2-substituted-5-{2-[(2-halobenzyl)thio)phenyl}-
1,3,4-oxadiazoles as anticonvulsant agents. Sci Pharm 76:185–201
Zhang P, Zhang X, Brown J, Vistisen D, Sicree R, Shaw J, Nichols G
(2010) Global healthcare expenditure on diabetes for 2010 and
2030. Diabetes Res Clin Pract 87:293–301
Bhattarai BR, Kafle B, Hwang JS, Khadka D, Lee SM, Kang JS, Ham
SW, Han IO, Park H, Cho H (2009) Thiazolidinedione deriv-
atives as PTP1B inhibitors with antihyperglycemic and antiobe-
sity effects. Bioorg Med Chem Lett 19:6161–6165
Cheon HG, Kim SM, Yang SD, Ha JD, Choi JK (2004) Discovery of
a novel protein tyrosine phosphatase-1B inhibitor, KR61639:
potential development as an antihyperglycemic agent. Eur J
Pharmacol 485:333–339
Cho SY, Baek JY, Han SS, Kang SK, Ha JD, Ahn JH, Lee JD, Kim
KR, Cheon HG, Rhee SD, Yang SD, Yon GH, Pak CS, Choi JK
(2006) PTP1B inhibitors: Cyclopenta[d][1,2]-oxazine deriva-
tives. Bioorg Med Chem Lett 16:499–502
Dixit M, Tripathi BK, Tamrakar AK, Srivastava AK, Kumar B, Goel
A (2007) Synthesis of benzofuran scaffold-based potential
PTP1B inhibitors. Bioorg Med Chem 15:727–734
Douty B, Wayland B, Ala PJ, Bower MJ, Pruitt J, Bostrom L, Wei M,
Klabe R, Gonneville L, Wynn R, Burn TC, Liu PCC, Combs AP,
Yue EW (2008) Isothiazolidinone inhibitors of PTP1B contain-
ing imidazoles and imidazolines. Bioorg Med Chem Lett
18:66–71
Egede LE, Ellis C (2010) Diabetes and depression: global perspec-
tives. Diabetes Res Clin Pract 87:302–312
Forghieri M, Laggner C, Paoli P, Langer T, Manao G, Camici G,
Bondioli L, Prati F, Costantino L (2009) Synthesis, activity and
Zhao H, Liu G, Xin Z, Serby MD, Pei Z, Szczepankiewicz BG,
Hajduk PJ, Bad-Zapatero C, Hutchins CW, Lubben TH, Ballaron
SJ, Haasch DL, Kaszubska W, Rondinone CM, Trevillyan JM,
Jirousek MR (2004) Isoxazole carboxylic acids as protein
tyrosine phosphatase 1B (PTP1B) inhibitors. Bioorg Med Chem
Lett 14:5543–5546
123